A large group of cell surface proteins of gram-positive bacteria are covalently anchored through their C-termini to the cell wall peptidoglycan. Most of these proteins are essential for pathogenic bacteria to establish successful infection of host tissues, and hence they are considered virulence factors.
Functionally, these surface proteins may be divided into three major groups: viz., 1) those with adhesin or invasion function (Patti et al. (1994) Annu. Rev. Microbiol. 48:585-617; Courtney et al. (1994) Infect. Immun. 62: 4868-4873) those with antiphagocytic activities (Fischetti et al. (1995) Infect. Immun. 63:149-153; Gigli et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76: 6596-6600), and 3) those that are enzymes that degrade surface components of host cells, thereby facilitating spread, and enzymes that hydrolyze large molecules in the surroundings into utilizable nutrients (Igarashi et al. (1995) Microbiol. Immunol. 39:853-860; Berry et al. (1996) J. Bacteriol. 178:4854-4860).
A striking feature of all these functionally and structurally diverse surface proteins is that they all possess a carboxy-terminal LPXTG sequence (Fischetti et al. (1990) Molec. Microbiol. 4: 1603-1605) which is cleaved during surface translocation at the septum (Mazmanian et al. (1999) Science 285: 760-762), resulting in a covalent linkage to cell wall peptidoglycan. In all cases, the genes for these proteins contain additional nucleotide sequences following that which encodes the LPXTG. These additional sequences encode a stretch of hydrophobic amino acids and positively charged C-terminal amino acids. Pancholi and Fischetti observed that the hydrophobic and positively charged amino acid sequences are missing in the cell wall-linked M protein, indicating that the precursor of M protein was cleaved at a site within or immediately C-terminal to the LPXTG sequence (Pancholi et al. (1988) J. Bacteriol. 170: 2618-2624). These findings strongly indicated that surface proteins become anchored to cell wall by a common mechanism (Fischetti et al. (1990) Molec. Microbiol. 4: 1603-1605).
Subsequently, it was also shown that deletion of either the LPXTG, or hydrophobic amino acid sequence or charged terminal amino acid from the precursor of protein A of S. aureus results in failure of protein A anchoring to the cell wall (Schneewind et al. (1992) Cell 70: 267-281), indicating that these sequences were essential for cell wall-anchoring process of these proteins. Collectively, these sequences are considered to be a cell wall sorting signal, which have now been shown to be present in over 100 surface proteins of gram-positive bacteria (Navarre et al. (1999) Microbiol. Molec. Biol. Reviews 63: 174-229; Pallen et al. (2001) Trends Microbiol. 9: 97-102).
Schneewind and colleagues have shown that the peptide bond between threonine and glycine of the LPXTG sequence of protein A becomes cleaved by an enzyme termed sortase, after which the carboxy-terminus of threonine becomes covalently attached to the amino group of one of the glycines of the pentaglycine cross bridge of the S. aureus cell wall (Schneewind et al. (1995) Science 268: 103-106). Recently, they have shown that S. aureus mutants defective in the anchoring of surface proteins to the cell wall carry a mutation in srt gene (Mazmanian et al. (1999) Science 285: 760-762). Subsequently they cloned the srt A gene in E. coli, and purified recombinant sortase. In vitro, the purified sortase cleaved the LPXTG sequence after threonine (Ton-That et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 12424-12429), and also covalently attached the surface protein with C-terminal LPXT to a triglycine substrate (Ton-That et al. (2001) J. Biol. Chem.). These results indicate that sortase possesses two functions, a specific endopeptidase and a transpeptidase. In addition, they showed that S. aureus mutants lacking sortase are unable to display surface proteins and are defective in establishing infection (Mazmanian et al. (2000) PNAS 97:5510-5515). An analysis of the genome of several gram-positive bacteria revealed that there are more than one sortase gene per bacterial genome (Pallen et al. (2001) Trends Microbiol. 9: 97-102).